Background binding of proteins, carbohydrates, cell lysates and the like to glass or other substrates employed in microarrays which include microspots containing protein capture agents or the like has posed a problem for a number of years. Non-specific binding of proteins to a microarray substrate increases the background noise when the microarray is imaged or the signals generated on the microspots are otherwise read; this makes it difficult to detect and distinguish signals being obtained from labels which should be specifically bound to particular spots, particularly in instances where the signal is relatively weak, because such background noise interferes and prevents obtaining precise readings.
To date two of the more common methods being used to attempt to alleviate or mitigate this problem involve manners of blocking the regions of the surface of the substrate surrounding each of the plurality of microspots. Such blocking has been done by chemically coating the surface of the substrate, e.g., by carrying out chemical reactions with the amino groups with which the glass surface has often been derivatized, as for example using succinic anhydride. A second method has employed the attachment of small molecules to the glass surface, for example, BSA, tRNA, skim milk solids, casein and the like. Various of these blocking methods are described in U.S. Patent Publication No. 2003/004823 which itself proposes the use of a “spreading enhancer solution” that would presumably be effective in assays employing nucleic acid probes. U.S. Pat. No. 5,248,595 is concerned with assays which employ antibodies, and it suggests removing uncomplexed protein materials using an aqueous wash, buffered to pH 9 or above and containing a specific anionic surfactant. U.S. Pat. No. 5,656,504 is concerned with assays employing antibodies, and it discloses coating glass supports with a layer of carboxylated dextran to prevent the undesired binding of proteins. U.S. Pat. No. 5,017,559 suggests blocking non-specific absorption of antibodies or the like onto a substrate by the use of milk protein plus a specific percentage of an organic acid.
Although various of these proposed solutions to the problem have shown some success, none of them has been widely accepted as the total solution to this problem. Accordingly the search has gone on for better solutions for combating background noise in reading microarrays, and particularly for those which use three-dimensional (3-D) hydrogel microspots.